Cervical cancer is the second most common cause of cancer deaths in women worldwide, with an incidence about a half million new cases cumulatively resulting in about a quarter of a million deaths every year. In the US, cervical cancer mortality rate has decreased substantially as a result of cervical cancer screening programs that detect precancerous conditions so they can be treated before developing into cancer. The current paradigm for cervical cancer screening is based on the Pap test, a cytologically-based test of cells scraped from the cervix and examined microscopically to detect changes indicating dysplastic cell growth. The test is subjective with significant inter-observer variability, and is limited by low sensitivity and high false positive results. Reports of false-negative rates in cervical cytology have varied widely, from as low as 1.6% to almost 28%. About 4 million abnormal Pap tests are diagnosed in the United States each year as atypical squamous cells of undetermined significance (ASC-US), atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion (ASC-H), low-grade squamous intraepithelial lesion (LSIL), or atypical glandular cells (AGC).
Under current practice guidelines, these cases are referred for colposcopy to further identify the subset of patients that will have clinically significant high-grade lesions (CIN2/3) or endocervical neoplasia on cervical biopsy. By some reports, patients with a cytologic diagnosis of ASC-US (over 2 million cases annually in the US) have only 5% to 17% chance of underlying CIN2/3 on cervical biopsy, and in LSIL (about 1.6 million cases in the US annually), CIN2/3 was found in up to 25%. These data suggest that for about 3 million cases with ASC-US or LSIL on Pap, colposcopy is unnecessary. Although colposcopic biopsy has historically been considered the gold standard, recent reports indicate that cervical biopsies may miss 33% to 50% of high-grade disease because of sampling or diagnostic errors. It therefore may be difficult to differentiate between false positive cervical cytology results and false-negative biopsy results. Therefore, there is strong need for a test to identify high-grade dysplasia to triage patients who can benefit most from intervention.
Although most low grade cervical dysplasias spontaneously regress without ever leading cervical cancer, dysplasia can serve as an indication that increased vigilance is needed. CIN1 is the most common and most benign form of cervical intraepithelial neoplasia and usually resolves spontaneously within two years. Because of this, LSIL results can be managed with a simple “watch and wait” philosophy. However, because there is a 12-16% chance of progression to more severe dysplasia, the physician may want to follow the results more aggressively by performing a colposcopy with biopsy. If the dysplasia progresses, treatment may be necessary. Therefore, what is needed is a method to detect HPV oncoproteins in situ. It would be particularly helpful in ASC-US or LSIL, or CIN1 patients to detect high-grade dysplasia cells and to identify those underlying CIN2 or above who may benefit immediate intervention, and avoid the anxiety associated with the “wait and see” approach.
Infection of specific epithelium cells by Human Papillomaviruses (HPV) and the resulting epithelial proliferation plays an important role for cervical carcinogenesis. About 99 percent of confirmed cervical cancer cases are found to be associated with HPV infection with biopsy-confirmed squamous intraepithelial lesions (SIL) or cervical intraepithelial neoplasia (CIN). The incidence of HPV infection, primarily transmitted through sexual contact, is highest among young women and about 20 million sexually-active men and women worldwide are currently infected. Approximately 1% of the population has genital warts and 4% of women have cervical precancerous lesions, such as low grade of squamous intraepithelial lesion (LSIL) or high grade of squamous intraepithelial lesion (HSIL) or atypical squamous cells of undetermined significance (ASC-US).
These lesions, preferentially observed in women aged 35-40 yrs, are associated with a high risk of progression toward invasive cervical cancer. It is generally thought that persistent HPV infection is essential for developing precancerous epithelial lesions. However, LSIL does not invariably progress to HSIL in women infected with a high-risk HPV strain. In fact, remission occurs in majority of human subjects diagnosed with LSIL. Although 99.7% of cervical cancers are HPV positive, viral genome integration into the host genome is required to facilitate expression of genes triggering development of HSIL or cancer. In fact, only one in every 10 women with persistent HPV infection develop higher grade CIN lesions, such as cervical intraepithelial neoplasia (CIN) grade 2 and grade 3 (CIN2, and CIN3, respectively), which in some cases, ultimately progress into cervical cancer.
Disease stages caused by HPV infection include an early stage HPV infection, a late stage HPV infection, Atypical squamous cells of undetermined significance (ASC-US), Atypical squamous cells, cannot exclude HSIL (ASC-H), Atypical glandular cells (AGC), low grade of squamous intraepithelial lesion (LSIL), high grade of squamous intraepithelial lesion (HSIL), cervical intraneoplasm CIN1, CIN2, CIN3 representing a mild, moderate, or severe cell dysplasia respectively, invasive cervical cancer, adenocarcinoma, or squamous cell carcinoma (SCC).
Nucleic acid-based HPV detection assays have been developed, but are not ideal for prognosing disease risk, in view of high cost, assay operation procedures, the requirements for facility, equipment, and highly trained personnel, and low positive predictive value for CIN. Current DNA-based assays cannot differentiate LSIL from HSIL, nor CIN lesions from non-transforming latent or remissive viral infection. Current mRNA-based assays for E6/E7 mRNA have approximately equivalent sensitivity to HPV DNA testing with higher positive predictive value. There are limited reports of assays to detect E6/E7 oncoproteins in situ. Longworth, M. S., and Laimins, L. A. (2004) Pathogenesis of Human Papillomaviruses in Differentiaing Epithelia, Microbiology and Molecular Biology Reviews 68, pp 362-372; and Tungteakkhun, S. S., and Duerksen-Hughes, P. J. (2008) Cellular Binding Partners of the Human Papillomavirus E6 Protein, Arch. Virol. 153, pp 397-408. What is needed is a low cost, simple, sensitive and specific assay that can be performed on routine practice of a clinical lab or doctor office and capable of detecting early stage of epithelial lesions, distinguish LSIL from HSIL, or predicting the risk of progression into cervical cancer.
Known protocols for the production of monoclonal antibodies to HPV are generally unsuitable for the production of anti-HPV monoclonal antibodies and cannot be used in immunocytochemical diagnostic tests for screening general human population. Veress, G., Konya, J., Csiky-Meszaros, T., Czegledy, J., and Gergely, L. (1994) Human Papillomavirus DNA and Anti-HPV Secretory IgA Antibodies in Cytologically Normal Cervical Specimens, Journal of Medical Virology 43, pp 201-207; Sun, Y., Shan, K. V., Muller, M., Munoz, N., Bosch, X. F., and Viscidi, P. P. (1994) Comparison of Peptide Enzyme-Linked Immunisorbent Assay and Radioimmunoprecipitation Assay with In Vitro-Translated Proteins for Detecti on fo Seruym Antibodies to Human Papillomavirus Type 16 E6 and E7 Proteins, Journal of Clinical Microbiology 1994, pp 2216-2220; Meschede, W., Zumbach, K., Braspenning, J., Scheffner, M., Benitez-Bribiesca, L., Luande, J., Gissmann, L., and Pawlita, M. (1998) Antibodies against Early Protein of Human Papillomaviruses as Diagnostic Markers for Invasive Cervical Cancer, Journal of Clinical Microbiology, 475-480; Sehr, P., Zymbach, K., and Pawlita, M. (2001) A Generic Capture ELISA for Recombinant Proteins Fused to Glutathione S-Transferase: Validation for HPV Serology, Journal of Immunological Methods 253, 153-162; Matlashewski, G., Banks, L., Wu-Liao, J., Spence, P., Pim, D., and Crawford, L. (1986) The Expression of Human Papillomavirus Type 18 E6 Protin in Bacteria and the Production of Anti-E6 Antibodies, J. Gen. Virol. 67, 1909-1916. This may reflect the use of recombinant proteins refolded following treatment with denaturants as immunogens for antibody production. Such antibodies react poorly with epitopes presented by native-conformation HPV protein produced by infected human cells. Additionally, epitopes recognized by prior art antibodies may be altered by standard procedures involved in the sampling, fixing and storing of clinical specimens. Other attempts to detect the presence of HPV related antibodies or viral proteins in a human subject by ELISA (enzyme linked immunoabsorbent assays) also generally lead to extremely low assay sensitivity and thus cannot be developed into a commercially suitable diagnostic test. Most of these ELISA assays target a single viral protein or short peptide fragments, which are not able to interact well or bind strongly and specifically to antibodies from the human subject. Specificity and sensitivity of such assays are so low that even using samples from patients confirmed with HPV associated invasive cervical cancer, only 53% of the patient samples were found positive for HPV infection. Nindl, I., Benitez-Bribiesca, L., Berumen, J., N, F., Fisher, S., Gross, G., Lopez-Carillo, L., Muller, M., Tommasino, M., Vazquez-Curiel, A., and Gissmann, L. (1994) Antibodies against Linear and Conformational Epitopes of the Human Papillomavirus (HPV) Type 16 E6 and E7 Oncoproteins in Sera of Cervical Cancer Patients, Arch. Virol. 137, 341-353. Given the testing populations come from general screening, with or without low grade, or precancerous lesions, the sensitivity of the assay will be too low to apply for clinical practice. Thus, there is no successful ELISA assay available as a diagnostic tool for clinical samples.
There are only about 15 types out of more than 100 types of HPV variants or strains that are associated with high-risk of CIN or cervical cancer risk. Also, around 70% of cervical cancer cases and 50% of CIN2 and CIN 3 cases are attributed to high-risk HPV type-16 and HPV type-18 infections. However, some progressive cervical cancer cases are related to infection by low risk HPV types, while infection of some HPV types will never progress into cervical cancer. It becomes important to identify those HPV infections and monitor expression of their particularly oncogenic proteins rather than just identify high risk type(s) of HPV infection. Thus, there is a need for detecting HPV oncoproteins as cervical cancer biomarkers to better identify the risk for developing HSIL, other precancerous lesions, or established cervical cancers.
Developing appropriate assays, such as HPV immunoassays, is needed for detection of such HPV oncoproteins or biomarkers for cervical cancer. The presence of E6/E7 oncoproteins in CIN 2 and CIN3 lesions can provide evidence indicating high risk of progression. However, prior art antibodies have limited utility for detecting E6/E7 oncoprotein in situ. M. S., and Laimins, L. A. (2004) Pathogenesis of Human Papillomaviruses in Differentiaing Epithelia, Microbiology and Molecular Biology Reviews 68, pp 362-372; and Tungteakkhun, S. S., and Duerksen-Hughes, P. J. (2008) Cellular Binding Partners of the Human Papillomavirus E6 Protein, Arch. Virol. 153, pp 397-408. Therefore, there is a need to develop antibodies and immunological assays for detecting HPV oncoproteins as cervical cancer biomarkers to identify HSIL or ≧CIN2 (CIN2 and above), or other precancerous lesions for use in screening for invasive cervical cancer and/or assessing the risk for malignant transformation into cervical cancer and other HPV associated cancers.